CD1d-restricted T cells (or "iNKT cells") have been reported to regulate an extremely diverse set of immunologic responses and diseases. Dysfunction of these T cells is clearly correlated with the development of autoimmunity, and in particular autoimmune diabetes. Despite the importance of CD1d-restricted T cells in this disease, how these T cells function normally and the exact nature of the disease-associated defects remains unclear. In this regard, potential regulatory functions that would be predicted to have significant impact on type 1 diabetes include recently described critical interactions of iNKT cells with dendritic cells and the activation-induced secretion of Th1 and Th2 cytokines. Th2 cytokine secretion by iNKT cells has been associated with protection from autoimmune diabetes in murine models. Conversely, iNKT cells cloned from patients with type 1 diabetes were found, amongst other defects, to have an extreme Th1 cytokine bias. In normal human volunteers, the CD4+ subset is responsible for Th2 cytokine production in vivo, whereas the CD4- (or "DN") subset was strongly biased towards the secretion of Th1 cytokines and cytotoxic function. Interestingly, individuals at risk for type 1 diabetes have a significant increase in the DN subset. Hence, CD4+ iNKT cells might serve to prevent progression to diabetes whilst DN iNKT cells might promote pro-inflammatory responses. A major hypothesis of the proposal is that the ratio of CD4 to DN iNKT cells is skewed toward DN iNKT cells. In addition, a bias towards proinflammatory DN iNKT cells and the a failure of CD4+ iNKT cells to up regulate Foxp3 and potentially other immunoregulatory genes might contribute to the progression to type 1 diabetes. To determine how iNKT cell subsets differentially signal using the same highly evolutionarily conserved TCR/CD1d antigen presentation system, we propose to develop proteomic methodology that will allow for the quantitative determination of site-specific phosphosignaling events in the following aim: Specific Aims: 1a. Analyze the requirements for the preferential activation of Foxp3 in CD4+ iNKT cells and determine the mechanism by which CD4+ iNKT cells exert immunoregulatory function. 1b. Determine phosphorylation and cytokine secretion patterns of CD4+ and DN iNKT cells. Type 1 diabetes is an autoimmune disease that results in destruction of pancreatic [unreadable] cells that produce insulin. This autoimmune disease affects as many as 1 in 300 persons in the United States. Despite advances in disease management and in the insulin preparations used, life span is shortened by 10-15 years in those developing type 1 diabetes under the age of 30. Microvascular and macrovascular complications occur in 30-50% of type 1 diabetes patients, and health care expenditures for diabetes alone in 2005 were estimated to have been in excess of $120 billion. Type 1 diabetes accounts for a disproportional share of such expenditures (in comparison to type 2 diabetes). The disease is recognized as a leading cause of blindness (retinopathy), heart disease, peripheral vascular disease, renal failure, and impotence. Therefore, an approach that involves early detection of disease activity, as well as a method of preventing pancreatic [unreadable] cell destruction such as activation of iNKT cells is appealing. [unreadable] [unreadable] [unreadable]